This proposal deals with aspects of the molecular biology of avian retroviruses relating to the mechanism of oncogenic transformation and to the control of endogenous retrovirus gene expression. In the first part experiments are described which focus on the proteins encoded by the myc oncogene. The development of an anti-peptide serum reactive with myc-encoded proteins has now allowed the identification and localization of these proteins to the cell nucleus. Use of this antiserum will allow us to carry out a detailed comparative analysis of the structure and subnuclear localization of myc proteins encoded by acute leukemia viruses, by rearranged myc genes in bursal lymphomas, and by the c-myc gene in normal avian cells. This will be achieved by different peptide and phosphopeptide mapping procedures using HPLC and two dimensional methods. Subnuclear fractionation studies will deal with separation of the components of the nuclear matrix and lamin complex. Crosslinking methods will be used to determine the nature of the associations between myc and other protein and DNA sequences present in the subnuclear fractions. Attempts will be made to isolate and characterize these species. Experiments aimed at elucidating the mechanism of myc induced transformation will involve cell cycle analysis of myc transformants using flow cytometry to relate the synthesis and compartmentalization of myc proteins to alterations in the cell cycle. A test of the activation in trans of host promoters by myc will be made using cloned genes injected into the frog oocyte. The effect of amplifying the levels of c-myc in a plasmid amplification vector will be assayed in terms of biological activity and localization and will aid in positive identification of the normal c-myc protein. The second part of this application relates to our findings on the control of endogenous retrovirus expression by DNA methylation. We propose to examine the generation of new recombinant viruses produced after the induction of defective viral genomes with azacytidine. We also plan to determine whether specific DNA sequences and specific events in spermatogenesis are involved in determining the methylation patterns found for retroviral genes. As a means of elucidating the nature of methylation we will prepare monoclonal antibodies against affinity purified eukaryotic methylatransferase as a first step in their characterization and molecular cloning.